Methods to increase the productivity of meterologous gene expression

ABSTRACT

The present invention provides a method of increasing heterologous protein expression/production in a cell by controlling proteolysis in the post ER compartment of the cell Proteolysis can be controlled by limiting/preventing export of proteins from the ER to the post ER compartment of the cell and/or by re-directing proteins from the vacuolar sorting route back to the ER or on towards the cell surface.

The present invention relates to a method of increasing heterologousprotein production in cells, cells which have been adapted to increaseheterologous protein production and uses of such cells.

BACKGROUND TO THE INVENTION

Protein synthesis and secretion by the secretory pathway occurs at theendoplasmic reticulum (ER) which maintains high levels of solubleresidents such as the lumenal binding protein (BiP), protein disulfideisomerase or calreticulin. The concentration of non-residents in the ERlumen which are in transit to other compartments, such as vacuoles orthe extracellular matrix, is usually much lower. Despite this, export ofnon-resident proteins from the ER is efficient while the cells are ableto restrict the leakage of the far more abundant ER residents to aminimum. In spite of significant advances in our understanding of themechanisms underlying vesicle budding and transport between the ER andthe Golgi, considerably less is known about the sorting of soluble cargomolecules during ER export.

The first model for protein secretion was inspired by the fact thatdifferent secreted proteins are secreted at various rates which impliedthat active export signals with different affinities for an ER exportreceptor exist. An alternative model was later proposed in which sortingsignals were envisaged to have a role as retention signals to deviateproteins from a default route that leads to secretion (Wieland et al.,1987). More recently (Barlowe et al., 1994) it has been observed thatCOPII vesicles, which carry out anterograde transport between the ER andthe Golgi, were found to be enriched for secretory cargo but lacked theER resident protein BiP. This observation indicated that secretory cargois selected and concentrated into COPII vesicles during budding so thatER residents are excluded. Further evidence for the concentration ofsecretory cargo during export was provided by the identification of adi-acidic ER export signal (Asp-X-Glu, where X is any amino acid) in thecytosolic tail of vesicular stomatitis glycoprotein (Balch et al., 1994;Nishimura and Balch, 1997).

These results suggest the presence of an export receptor. However,identification of an export signal on any soluble protein remainselusive to date.

Eukaryotic cells of fungi, yeasts, algae, plants, insects and mammalsall share the same secretory pathway consisting of the endoplasmicreticulum, the Golgi apparatus and lytic (vacuolar) compartments. Plantcells contain at least two functionally distinct vacuolar compartments,the central lytic vacuole which is related to the mammalian lysosome,and the so-called storage vacuoles, which appear to be unique to theplant kingdom. Whereas the lytic vacuole is typical for vegetativecells, storage vacuoles are mostly found in reserve tissues of seeds.Exceptions to this are the vegetative storage vacuoles which are formedduring stress conditions and which could be related to a neutralvacuolar compartment recently discovered in tobacco protoplasts. It wasinitially thought that the various vacuolar compartments share a commonorigin but change appearance and contents according to the physiologicalconditions or tissue type. However, the simultaneous presence of storagevacuoles and lytic vacuoles within the same cell, as determined usingspecific membrane markers, argues against this. In addition, the greatvariety of vacuolar sorting signals described in plants also points atthe possibility that the various types of vacuoles have a differentorigin and are supported by different protein transport pathways.

It is apparent that there are many factors involved in ER export andvacuolar sorting which are are yet to be identified and which may revealfurther unique features of the plant secretory pathway. To date, asingle putative vacuolar sorting receptor has been identified in plants(BP80 or VSR_(PS-1)) and tested either by in vitro ligand binding assays(Kirsch et al., 1994) or via re-constitution in a heterologous in vivosystem using yeast (Humair et al., 2001).

It is believed that disposal of proteins may be as a result of a“quality control” mechanism in the secretory pathway, which mayrecognise heterologous proteins as unsuitable for secretion and signalstargeting to the possible disposal sites, which comprise the lyticvacuole or the cytosolic proteasome. The present invention provides amethod to purposefully prevent such disposal from occurring.

Heterologous protein production using the secretory pathway of plantsand/or micro-organisms is a potentially commercially valuable method ofproducing, for example, mammalian serum proteins. However a problem withsuch a method is that the secretion of heterologous proteins is oftenlow-yielding which has thus prevented heterologous protein production bythe plant secretory pathway to enter into widespread industrialuse/applications.

Attempts have been made to increase the yield of proteins produced bythe secretory pathway, which have been disappointing. Prior art methodshave attempted to solve the problem of low yields by increasing the rateof synthesis. The solution provided by the present invention is thusinnovative compared to the teachings in the prior art.

A method which could improve on prior art performance and increase theyield of heterologous protein production in plant and/or microbial cellswould offer immediate advantage to the art.

Moreover, such a method would be applicable to the large scaleproduction of many mammalian proteins and other proteins of high value.

STATEMENT OF THE INVENTION

According to the broadest aspect of the invention there is provided amethod of increasing heterologous protein expression/production by cellby regulating/controlling proteolysis, especially in the post ERcompartment of the cell.

The present invention is based on the unexpected observation thatproteolysis, and not synthesis, is a limiting factor in heterologousprotein production.

Reference herein to heterologous protein is intended to include anyforeign or non-native protein that is produced by the cell.

According to an aspect of the invention there is provided a method ofincreasing heterologous protein production in a cell comprising limitingand/or decreasing proteolysis by:

-   -   (i) limiting/preventing export of proteins from the ER to the        post ER compartment of the cell;    -   (ii) re-directing proteins from the vacuolar sorting route back        to the ER; and/or    -   (iii) re-directing proteins from the vacuolar sorting route to        the cell surface.

For strategy (i), the host cell can be of any eukaryotic origin, i.e.plant, fungal, yeast or mammalian origin. For strategies (ii) and (iii),the method is restrictive to plants but may be applied to othereukaryotic cells with or without minor modifications.

According to a further aspect of the invention there is provided a celladapted so that proteolysis is limited and/or decreased.

Preferably the cell is adapted by the method of the present invention.

According to a yet further aspect of the invention there is provided useof the cell/cells of the present invention in increasing the productionof a heterologous protein.

In one embodiment of the invention, the cell contains heterologous DNAencoding a mammalian serum protein. This particular embodiment whenapplied to plant, fungal or bacterial cells is particularly advantageousin that the risk of pathogen-contaminated blood products, such ascontamination with hepatitis and/or HIV, is mitigated. It is envisagedthat serum proteins produced by the method of the present invention willprovide an alternative and safe source of donor serum and blood factors.

In other embodiments of the invention the cells contain heterologous DNAencoding, for example and without limitation, hormones such as humangrowth hormones and endocrine products such as insulin and thyroxine. Aspreviously mentioned production of such hormones by the method of thepresent invention will provide a pathogen free product, especially whenapplied to production by to plant, fungal or yeast cells.

It is believed that proteolysis occurs in the lytic vacuole of plants orthe central vacuole of micro-organisms such as fungi or yeasts. Thepresent invention relates to a method to overcome or limit proteolysisby either preventing export of proteins out of the ER and/or byredirecting proteins from the vacuolar sorting route back to the ER orthe cell surface, and in this way the yield of heterologous proteinexpression is increased.

We provide evidence that prevention of ER export leads to drasticincreases in the total yield of protein, without the requirement ofincreasing the synthesis rate of the protein. We have shown increases inyield which are not marginal but surprisingly can be up to a hundredfold. This result is most surprising, because it involves the disruptionof an essential biochemical pathway which would not be expected to bebeneficial to an organism.

Preferably, limiting/preventing export of proteins out of the ER is byinhibition of COPII transport. This type of ER export is conserved inall eukaryotic cells and thus represents a universal target forinhibition.

Preferably, inhibition of COPII transport is achieved by either of thefollowing methods:

-   -   (i) inhibition of COPII dependent vesicle budding via        overproduction of the Sar1-specific guanosine exchange factor        Sec 12;    -   (ii) co-expression of a mutant GTPase Sar1 which is less        sensitive to its GTPase activating protein and as a result is        defective in GTP hydrolysis.

Preferably, the exchange factor is Sec12p or an isoform thereof.

Preferably, the mutant GTPase is Sar1 or ARF1 or isoforms thereof.

Both proteins have equivalents in other eukaryotic cells. Plant Sec 12and Sar1 homologs are so similar to their yeast counterparts that theycan functionally complement the yeast homologs (d'Enfert et al., 1992).

Preferably, re-directing proteins from the vacuolar sorting route backto the ER may be achieved by co-expressing a modified vacuolar sortingreceptor which carries an engineered ER retention signal and which istransported from the Golgi apparatus back to the ER instead of ontowards the vacuolar compartment.

For example, in plants preferably the receptor is BP80 or a closeisoform of said receptor and preferably the engineering of the ERretention signal is such that it does not interfere with its ligandbinding properties.

In other eukaryotic cells, vacuolar sorting receptors may be modified insimilar ways to achieve the same effect. The closest relative in yeastto BP80 is thought to be the VPS10 gene product and also mammalian cellscontain receptor proteins which bind to ligands in the Golgi apparatusand which initiate targeting to the vacuole. It will thus be appreciatedthat any modified receptor molecule, modified in such a way as tore-direct proteins back to the ER instead of directing them to the lyticcompartment are included within the scope of the present invention.

In one embodiment of the invention, the cell is adapted so that the BP80phenotype is depleted by producing a BP80-mycHDEL receptor. We havefound that BP80-mycHDEL is capable of retrieving proteins destined forthe vacuole.

In an alternative embodiment of the invention BP80 is specificallyengineered so as to reach the cell surface with its protein cargo, sothat the protein may be secreted at the cell surface.

Preferably, the method of re-directing vacuolar proteins to the cellsurface is via co-expression of a GTP-restricted form of the lowmolecular weight GTPase ARF1. For example, in plants, suchover-expression causes transient secretion of those vacuolar proteinsdestined to the lytic vacuole via the BP80-route. If applied at thecorrect concentration and prior to harvesting of cells, such treatmentwill re-direct a significant proportion of the proteins normallydestined to the lytic vacuole towards the cell surface and theextra-cellular fluid, in which many heterologous proteins would bestable. Purification from the culture medium would be easier, andcombined with the higher stability and yield of the gene product thiswould constitute an immediate advantage.

Preferably, cells containing heterologous DNA are incubated inappropriate fermentation/incubation media for sufficient time togenerate enough cell mass, subsequently the cells are subjected toeither one or a combination of the previously described proteolysisinhibition treatments prior to harvesting of the heterologous protein.

Typically cells are subjected to either one or both of the proteolysistreatments for 12 to 48 hours prior to harvesting, but this may dependon the gene-product and should not be regarded as an exclusiverecommendation or a limiting factor of the method of the invention.

It will be appreciated that other methods that control/regulateproteolysis are equally applicable and are intended to be included inthe scope of the present application. For example, and withoutlimitation, the disposal of malfolded proteins after retrogradetransport from the ER to the cytosolic proteasome, which has been welldescribed in mammals and yeast, may also be expected to operate inplants in addition to the vacuolar degradation. For instance, ifprevention of ER export does not stabilise the heterologous protein inquestion (Example 1), retrograde translocation to the cytosolicproteasome could be prevented via mutant Sec61 overexpression.Similarly, vacuolar transport could also be re-directed to the cellsurface instead of the endoplasmic reticulum (Examples 2 and 3).

Numerous ways of manipulating the secretory pathway could be envisagedto limit proteolysis. The invention is based on the surprising discoverythat proteolysis and not synthesis is the limiting factor inheterologous protein production. Therefore, the three examples givenshould not be seen as an exclusive list of approaches.

Preferably, the method of the present invention will be utilised forlarge scale production of heterologous proteins. It is envisaged thatplant/fungal/yeast/mammalian cells containing appropriate heterologousDNA encoding the protein of choice will be incubated in fermentationvats under suitable conditions and that the heterologous protein will beharvested therefrom. It is believed that the method of the presentinvention will provide improved yields in heterologous proteinexpression and, when used to produce serum/blood proteins mitigateproblems associated with pathogen contaminated blood products.

Preferably, the method of the present invention further comprises anyone or more of the following steps:

-   -   (i) cell culture;    -   (ii) harvesting;    -   (iii) purification;    -   (iv) modification;    -   (v) formulation; or    -   (vi) lyophilisation.

It will be appreciated that some of the heterologous protein produced bythe present invention may be subjected to post synthesis modificationssuch as glycosylation or glycan modification.

Preferably, the formulation step includes providing the heterologousprotein in a suitable diluent, carrier or exipient or alternatively theproduct may be freeze dried/lyophilised for subsequent use.

According to a further aspect of the invention there is provided amethod of producing heterologous mammalian proteins comprising:

-   -   (i) incubating cells containing heterologous DNA encoding the        protein of choice in appropriate incubation media until        sufficient cell mass is generated;    -   (ii) reducing export of proteins out of the ER and/or        re-directing proteins from the vacuolar sorting route back to        the ER or the cell surface and;    -   (iii) harvesting the heterologous protein.

Preferably, the method further includes the step of disposal ofremaining cell debris containing genetically modified material forexample by fermentation to methane and/or incineration of solidmaterial.

The method of the present invention is thus advantageous in a totalcontainment strategy.

Preferably the method further includes any of the preferred featureshereinbefore described.

According to a yet further aspect of the invention there is provided amethod of producing heterologous mammalian serum proteins comprising:

-   -   (i) incubating cells containing heterologous DNA encoding the        serum protein of choice in appropriate incubation media media        until sufficient cell mass is generated;    -   (ii) reducing export of proteins out of the ER and/or        re-directing proteins from the vacuolar sorting route back to        the ER and;    -   (iii) harvesting the heterologous serum protein.

It will be appreciated that mammalian serum proteins is intended toinclude blood clotting factors, immunoglobulins and other such bloodcomponents.

Preferably, the method further includes any one or more of the featureshereinbefore described.

According to a yet further aspect of the invention there is provided aprotein product produced by the method of the present invention.

Preferably the method of producing the product further includes any ofthe preferred features hereinbefore described.

The invention will now be described by way of example only withreference to the following Figures wherein:

FIG. 1 illustrates Amy-HDEL secretion;

FIG. 2 illustrates Amy-HDEL secretion in stably transformed tobaccosuspensions;

FIG. 3 illustrates the effect of cargo dosage and the temperature onsecretion and retention;

FIG. 4 illustrates in vivo manipulation of COPII dependent ER export;

FIG. 5 illustrates evidence that Sar1 can restore Sec12 mediatedinhibition of secretion;

FIG. 6 illustrates evidence that Sar1p overexpression alone does notinfluence secretion;

FIG. 7 illustrates membrane recruitment of Sar1p by increasing thelevels of Sec12p;

FIG. 8 illustrates inhibition of ER-Golgi transport using mutant Sar1and comparison with Sec12;

FIG. 9 illustrates secretion of ER residents and bull flow markers isCOPII dependent;

FIG. 10 shows a comparison of steady state protein levels and the denovo synthesis rate;

FIG. 11 illustrates Example 1;

FIG. 12 illustrates transport of the vacuolar marker phytepsin;

FIG. 13 illustrates transport of soluble BP80 and HDEL tagged solubleBP80;

FIG. 14 shows that BP80-HDEL can cause specific accumulation ofun-processed phytepsin in the ER without affecting a non-ligand for BP80(α-amylase);

FIG. 15 illustrates Example 2;

FIG. 16 illustrates a model predicted transport routes;

FIG. 17 shows the influence of increasing BFA concentration on thetransport of amy and amy-HDEL in transient expression;

FIG. 18 shows the influence of BFA on the transport of amy and amy-HDELin stable transformants in function of the time;

FIG. 19 shows transient expression of recombinant ARF1p and ARF1(Q71L)pin tobacco protoplasts;

FIG. 20 shows the influence of Sec12, Sar1 and ARF1 on anterograde andretrograde transport;

FIG. 21 shows the influence of Sec12, Sar1 and ARF1 on the transport ofbarley phytepsin;

FIG. 22 shows the influence of increasing BFA concentration on thetransport of phytepsin;

FIG. 23 illustrates the propeptides of sweet potato sporamin and barleylectin are functional at the C-terminus of α-amylase;

FIG. 24 shows ARF1(Q71L)-induced secretion specifically occurs forsequence specific sorting signals; and

FIG. 25 shows ARF1(Q71L)-induced secretion can not be mimicked by BFAand is dependent on the NPIRL motif.

DETAILED DESCRIPTION OF THE INVENTION

With regard to mole detailed description of the Figures:

FIG. 1. Amy-HDEL Secretion is Due to Saturation of the HDEL Receptor.

Time course of Amy (left) and Amy-HDEL (right) expression, showingα-amylase activity in cells (open squares), in the culture medium (blacksquares) and the total activity (circles). The lower panel shows theratio between the extracellular and the intracellular α-amylaseactivity, termed secretion index (SI). Note the ten-fold difference inthe scales.

FIG. 2. Amy-HDEL Secretion in Stably Transformed Tobacco Suspensions:

Medium and cells analysed via Coomassie stained protein gels or Westernblot analysis. Control cells are untransformed, and amy producers arecompared with amy-HDEL producers. All cultures were analysed 1 weekafter inoculum (1) or two weeks after inoculum (2). Note that bothproteins are well recovered in the medium, but that the presence of HDELmainly results in an increase in the cellular levels.

FIG. 3. Effect of Cargo Dosage and the Temperature on Secretion andRetention

(A) Total activity and Secretion index of Amy (white bars) compared toAmy-HDEL (black bars) measured 24 hours after transfection ofprotoplasts. The concentration of the plasmids used for transfection isindicated. The SI of Amy is given on the left y-axis while the SI ofAmy-HDEL is given on the right y-axis.

(B) Influence of the temperature on protein synthesis (total activity)and the secretion index of Amy (white) compared to Amy-HDEL (black). TheSI of Amy is given on the left y-axis while the SI of Amy-HDEL is givenon the right y-axis.

FIG. 4. In Vivo Manipulation of COPII Dependent ER Export

Dosage dependent inhibition of secretion of Amy and Amy-HDEL by Sec12pco-expression measured 24 hours after transfection. The amount of Sec12pencoding plasmid is given in micrograms and a constant amount of Amy(white bars) or Amy-HDEL (black bars) encoding plasmids (2 μg) was usedin each lane. The SI of Amy is given on the left y-axis while the SI ofAmy-HDEL is given on the right y-axis. The western blot shows theexpression of Sec12p in each lane.

FIG. 5. Evidence that Sar1 can Restore Sec12 Mediated Inhibition ofSecretion

Partial reconstitution of Sec12p mediated inhibition of secretion byco-expression of Sar1p. Annotations are as in FIG. 4. The western blotillustrates the constant levels of Sec12p and the increased expressionof Sar1p (concentrations of Sar1 encoding plasmid given in μg).

FIG. 6. Demonstration that Sar1p Overexpression Alone does not InfluenceSecretion.

Annotations are as in B), but only the secretion of Amy was tested withmaximum levels of Sec12p and Sar1p.

FIG. 7. Membrane Recruitment of Sar1p by Increasing the Levels ofSec12p.

Protoplasts were extracted by osmotic shock to yield the cytoplasmicfraction (sol.) and the membrane fraction (mem.). Sec12p was onlypresent in the membrane fraction. Quantities of plasmids are given inμg. Note that at high expression levels of Sec12, Sar1 is recruited tothe membrane fraction at the expense of the cytosolic localisation.

FIG. 8. Inhibition of ER-Golgi Transport Using Mutant Sar1 andComparison with Sec12

(A) Transport assay to detect the influence of the dominant negativeGTP-trapped mutant of Sar1 after 24 hours of co-expression. The numbersabove the lanes refer to the amount in μg of plasmids encoding eitherSec12-, Sar1 wild type (Sar1p WT), or mutant Sar1 (Sar1p M). The upperpanel shows a protein gel blot to detect the three differentco-expressed proteins. The lower panel shows the secretion index andtotal activity respectively, corresponding to two independent transportassays using the secretory market α-amylase. Lanes are as in the upperpanel. Note the strong reduction in the secretion index when both Sec12and mutant Sar1 are co-expressed. Note also that Sar1p M alone causes areduction in the total yield of α-amylase, and that further inhibitionof secretion by superimposing increased Sec12 levels restores some ofthe yield. (B) Transport assay to monitor the effect of co-expressedwild type Sar1 on a constant level of mutant Sar1. Shown is thesecretion index and the lanes indicate the quantities of thecorresponding plasmids transfected in μg. Note that only a hundred foldexcess of wild type Sar1 rescues some of the secretion inhibited by themutant GTPase.

FIG. 9. Secretion of ER Residents and Bulk Flow Markers is COPIIDependent.

The culture medium (M) and the cells (C) of protoplast suspensions wererecovered 48 hours after transfection and cargo molecules were detectedeither by protein gel blots (upper panels) or via enzymatic analysis(lower panels). The numbers above the lanes refer to the amount ofSec12p encoding plasmid (Sec12) or mutant Sar1 encoding plasmid (Sar1M)in μg. As cargo molecules, the bulk flow marker PAT or CalreticulinΔHDEL(CalretΔ) were co-expressed in constant amounts (2 μg).CalreticulinΔHDEL was also 10-fold over-expressed (CalretΔ OE) in thebottom panel (20 μg). The control lane is devoid of any cargo molecule.Note that calreticulinΔHDEL secretion is only seen when the protein isoverexpressed. The lower panel shows the effect of the variousinhibitory levels of Sec12 and mutant Sar1 on the transport of thesecretory marker α-amylase. Shown is the secretion index, the totalactivity, and the activity in the cells (grey) and the culture medium(white) as indicated and lanes correspond to the upper panel. Note thatthe total α-amylase activity is slightly reduced with increasing levelsof either transport inhibitors. Note also that the intracellularaccumulation of α-amylase is not higher as that of PAT andcalreticulinΔHDEL.

FIG. 10: Comparison of Steady State Protein Levels and the De NovoSynthesis Rate.

Tobacco protoplasts were harvested 24 hours after transfection and equalportions were analysed for protein levels (Steady state) using proteingel blots, or the ability to synthesize protein de novo during a 30minute pulse labelling procedure (Pulse), detected via quantitativeimmunoprecipitation, subsequent SDS-PAGE and autoradiography. Thenumbers above the lanes refer to the amount of Sec12p encoding plasmid(Sec12) in μg. CalreticulinΔHDEL was co-expressed in low amounts (2 μg)as in FIG. 6 which does not allow detection of protein in the medium.Note that detected levels using pulse labelling were constant in sharpcontrast to the steady state protein levels.

FIG. 11: Illustration of Example 1.

Upon inhibition of COPII dependent ER export, the Golgi apparatus isexpected to desintegrate and transport to distal locations is inhibited,followed by accumulation of proteins in the ER. This results in dilatedER and presumably alternative transport to the storage vacuoles, whichhave been hypothesised to simply be the dilated ER itself.

FIG. 12: Illustration of Transport of the Vacuolar Marker Phytepsin

(A) Transport of phytepsin to the vacuoles results in processing,whereas secretion (due to saturation of the vacuolar transport route)results in the detection of un-processed protein in the culture medium.Inhibition of COPII transport by Sec12 overproduction increases theamount of unprocessed phytepsin in the cells and abolishes secretion ofthe unprocessed form, thus indicating that phytepsin exits the ERthrough the COPII route.

(B) Illustration of the processing steps associated with phytepsinsynthesis and transport.

FIG. 13. Transport of Soluble BP80 and HDEL Tagged Soluble BP80.

(A) Transient expression showing that secretion of soluble BP80 isprevented by HDEL tagging. (B) Illustration of HDEL tagged BP80 and itsinteraction with the HDEL receptor ERD2. (C) Illustration of howBP80-HDEL can be employed to recycle vacuolar proteins back to the ERinstead to the vacuoles via the dominant effect of the overproducedsoluble BP80 derivative.

FIG. 14. Demonstration that BP80-HDEL can Cause Specific Accumulation ofUn-Processed Phytepsin in the ER without Affecting a Non-Ligand for BP80(α-Amylase).

Transient expression of phytepsin and an increasing amount of eithersoluble BP80 or BP80-HDEL. Note that Secretion is abolished by BP80-HDELand that the un-processed form accumulates in the ER.

FIG. 15: Illustration of Example 2.

Upon overexpression of BP80-HDEL, proteins normally destined to thevacuoles will recycle from the Golgi back to the ER and accumulatethere. This results in dilated ER and presumably alternative transportto the storage vacuoles, which have been hypothesised to simply be thedilated ER itself.

FIG. 16: Model Illustrating Predicted Transport Routes

Schematic drawing illustrating current knowledge on defined transportsteps towards and from the Golgi apparatus in plants. COPII dependent ERexport (1) is balanced by retrograde COPII mediated transport (2),clathrin mediated transport (5) to the prevacuolar compartment ismatched by a retrograde route (6) which is believed to be dependent onthe retromer complex. Anterograde intra-Golgi transport (3) andtransport to the storage vacuole (SV) (4) and secretion (7) to theplasma membrane (PM) is mediated by as yet un-known transportmechanisms. Unknown transport pathways (?) denote export from the PVC,recycling from the SV, as well as unusually large ER export vessels(Hara-Nishimura et al., 1998; Toyooka et al., 2000) as well as COPIIindependent ER export of phytepsinΔPSI (Törmäkangas et al., 2001).

Cargo molecules used in this study and known sorting receptors of theplant secretory pathway are listed, including the predicted transportroutes followed. White numbers in dark field show transport routes onlyfollowed during saturation of receptor mediated routes. Amy-spo andamy-spoM represent α-amylase fusion proteins containing WT or mutated(M) forms of the sweet potato sporamin (Koide et al., 1997). Amy-blrepresents an α-amylase fusion protein with the propeptide of barleylectin (Bednarek and Raikhel, 1991). A non-functional derivative basedon the established C-terminal double glycin addition (Dombrowski et al.,1993) is denoted as amy-blGG. Since barley lectin and sweet potatosporamin were shown to be transported to the same kind of vacuole(Schroeder et al., 1993) via different sorting mechanisms (Matsuoka etal., 1995), it can not simply be postulated that amy-bl follows route 4.Therefore, the expected Golgi derived transport route is left open andis indicated by the symbol (?).

FIG. 17: Influence of Increasing BFA Concentration on the Transport ofAmy and Amy-HDEL in Transient Expression.

The upper panel shows the secretion index (SI) of amy (open bars) andamy-HDEL (grey bars) in function of increasing concentration ofBrefeldin A (indicated at the bottom in μg/ml final concentration). Theleft hand y-axis denotes the SI of amy, and the right hand axis denotesthe SI of amy-HDEL. Note the much stronger reduction of the SI for amycompared to amy-HDEL. The lower panel shows the total activity(medium+cells) from the same experiments in the upper panel, given inchange in OD per ml of protoplast suspension per minute. Note theslightly increased total activity for amy-HDEL when BFA is present.

FIG. 18: Influence of BFA on the Transport of Amy and Amy-HDEL in StableTransformants in Function of the Time.

The upper panel shows the SI of amy from washed protoplast suspensionsduring a 240 minute period after incubation in the presence (grey bars)and absence (open bars) of 30 μg/ml BFA. The lower panel shows the samefor amy-HDEL. Note that BFA inhibits secretion of amy, as seen by alower SI when compared to mock-treated cell suspension, whereas thisdifference is not seen for amy-HDEL.

FIG. 19: Transient Expression of Recombinant ARF1p and ARF1(Q71L)p inTobacco protoplasts.

Co-expression of the secretory marker α-amylase (amy) with either wildtype ARF1p or mutant ARF1(Q71L)p. The upper panel shows the SI for amyunder control conditions (con) in comparison with the co-expressed ARF1proteins. The lower panel shows the total amount of endogenous ARF1p(con) in comparison with the higher protein level when 60 μg of ARF1- orARF1(Q71L) encoding plasmid was electroporated. Note the profound effectof ARF1(Q71L)p on amy secretion in contrast to the wild type molecule.

FIG. 20: Influence of Sec12, Sar1 and ARF1 on Anterograde and RetrogradeTransport.

Co-expression of amy and amy-HDEL with increasing concentrations ofSec12-, Sar1(H76L)- or ARF1(Q71L)-encoding plasmids. The effectorplasmid concentrations are given below each lane. SI values for amy aregiven in open bars, values for amy-HDEL are given in grey bars. Standarderrors are indicated for all measurements. The left hand y-axis denotesthe SI of amy, and the right hand axis denotes the SI of amy-HDEL. Notethat only Sec12p over-expression causes efficient inhibition ofsecretion of amy-HDEL, whereas the two trans-dominant GTPase mutantsseem to inhibit mainly amy-secretion and have little effect on amy-HDELtransport.

FIG. 21: Influence of Sec12, Sar1 and ARF1 on the Transport of BarleyPhytepsin

Co-expression of the vacuolar protein phytepsin with increasingconcentrations of Sec12-, Sar1(H76L)- or ARF1(Q71L)-encoding plasmids.The effector plasmid concentrations are given below each lane. Equalvolumes of medium (M) and Cell (C) samples are compared by protein-gelblotting. The open arrow denotes the un-processed pro-phytepsin, whereasthe closed arrow denotes the lower molecular weight vacuolar form ofphytepsin devoid of the N-terminal pro-peptide (Törmäkangas et al.,2001). Note that ARF(Q71L) co-expression causes an induced secretion ofpro-phytepsin to the medium, whereas Sec12p and Sar1(H76L)p inhibit bothsecretion and vacuolar sorting.

FIG. 22: Influence of Increasing BFA Concentration on the Transport ofPhytepsin.

Transient expression experiment showing the influence of BFA (indicatedabove each lane in μg/ml final concentration) on secretion to the medium(M), intracellular retention (C) and the partitioning between processed(closed arrows) and un-processed (open arrows) forms of phyteposin. Notethat BFA inhibits both secretion and intracellular processing ofphytepsin.

FIG. 23: The Propeptides of Sweet Potato Sporamin and Barley Lectin areFunctional at the C-Terminus of α-Amylase.

Transient expression experiment showing the SI for amy, amy-HDEL,amy-spo, amy-spoM, amy-bl, and amy-blGG (see FIG. 1) Note that thatintracellular retention of amy-spo is almost complete with hardly anyevidence for saturation of the BP80-mediated sorting route, whereasamy-bl retention is leaky and comparable to that of amy-HDEL.

FIG. 24: ARF1(Q71L)-Induced Secretion Specifically Occurs for SequenceSpecific Sorting Signals.

Transient expression experiment testing the influence of an increasingconcentration of ARF1(Q71L)p on the transport of amy-spo and amy-bl. A)Extracellular (open bars) and intracellular (grey bars) activities(given in change in OD per ml of protoplast suspension per minute) shownfor amy-spo and amy-bl in function of increased dosage of ARF1(Q71L)p.The plasmid concentration is given below each lane in μg. Note anincrease in the extracellular level of amy-spo at high concentrations ofARF1(Q71L)p, in contrast to amy-bl whose secretion diminishes underthose conditions. B) Data from A) represented as SI or Total activity(change in OD per ml of protoplast suspension per minute) for directcomparison between amy-spo and amy-bl. Amy-spo is denoted in light grey(right hand y-axis for SI) and amy-bl is denoted in dark grey (left handy-axis for SI). Note the opposite behaviour of the SI for the two fusionproteins. Note also that the total amy-bl activity drops with increasingdosage of ARF1(Q71L)p while that of amy-spo increases slightly.

FIG. 25: ARF1(Q71L)-Induced Secretion can not be Mimicked by BFA and isDependent on the NPIRL Motif.

A) Transient expression experiment showing the influence of anincreasing concentration of ARF1(Q71L)p on the intracellular (grey bars)and extracellular (open bars) levels of amy-spoM (exhibiting a mutatedsorting motif). The plasmid concentration is given below each lane inμg. The first two lanes are a positive control demonstrating the abilityof ARF1(Q71L)p to induce secretion of the wild-type sporamin fusion.Note that none of the concentrations of ARF1(Q71L)-encoding plasmidleads to induced secretion of the mutant sporamin fusion (compare withFIG. 9A, upper panel). B) Transient expression experiment showing theinfluence of increasing concentration of BFA (indicated at the bottom inμg/ml final concentration) on the intracellular (grey bars) andextracellular (open bars) levels of amy-spo. Note that BFA does notinduce the secretion to the medium as seen for ARF1(Q71L)p co-expression(compare to FIG. 9A, upper panel).

Materials and Methods

Plasmid Construction for Transient and Stable Expression

All DNA manipulations were done according to established procedures. TheEscherichia coli MC1061 strain (Casadaban and Cohen, 1980) was used forthe amplification of all plasmids. Plasmids encoding for x-amylase andα-amylase-HDEL were described (Crofts et al., 1999) To allow detectionof transiently expressed calreticulinΔHDEL (Crofts et al., 1999) amongendogenous wild type calreticulin, pDE314C (Crofts et al., 1999) wasengineered to incorporate a c-myc tag just prior to the stop-codon,resulting in the plasmid pCalmyc. The Sec12 and Sar1 overexpressionplasmids were generated through PCR amplification of the correspondingcDNA clones (d'Enfert et al., 1992) to result in coding regions placedin between the Cauliflower Mosaic Virus 35S promoter and the 3′untranslated end of the nopaline synthase gene as in pDE314C (Crofts etal., 1999). Site-directed mutagenesis of the Sar1 coding region wascarried out to exchange the histidine codon in position 74 with aleucine codon, using the following two PCR primers: SARH74L-sense(5′TTGATTTGGGTGGTCTTCAGATTGCTCGTAG3′) SEQ ID NO:1 and SARH74L-anti(5′CTACGAGCAATCTGAAGACCACCCAAATCAA 3′) SEQ ID NO:2, yielding plasmidpLL18

Previously established plasmids were used encoding α-amylase andα-amylase-HDEL (Crofts et al., 1999), and phytepsin (Törmäkangas et al.,2001). The four plasmids encoding the α-amylase derivatives amy-spo(pSLH44), amy-spoM, amy-bl and amy-blGG were generated by insertingannealed oligonucleotide pairs between the BglII and XbaI sites of theα-amylase encoding plasmid which overlap with the last codon and thestop-codon. The following oligonucleotide pairs were used to generatethe C-terminally fused peptide encoding regions: spo-sense(5′GATCAGATTCAATCCCATCCGCCTCCCCACCACACA CTAACT3′) (SEQ ID NO 3:,spo-anti (5′CTAGAGTTAGTGTGTGGTGGGGAGGCGGATGGGATT GAATCT3′) (SEQ IDNO:4); spoM-sense (5′GATCAGATTCAATCCCGGTCG CGGTCCCACCACACACTAA CT3′)(SEQ ID NO:5); spoM-anti (5′CTAGAGTTAGTGTGTGGTGGGACCGCGACCGGGATTGAATCT3′) (SEQ ID NO:6); bl-sense(5′GATCGTTTTTGCTGAAGCTATTGCTGCTAATTCTACTCTTGCTTGCT GGATAAT3′), (SEQ IDNO:7); bl-anti (5′CTAGATTATTCAGCAACAAGAGTAGAATTAGCAGCAATAGCTTCAGCAAAAAC3′) (SEQ ID NO:8); blGG-sense(5′GATCGTTTTTGCTGAAGCTATTGCTGCTAATTCTACTCTTGTTGCTGAA GGTGGATAAT3′), (SEQID NO:9); blGG-anti (5′CTAGATTATCCACCTTCAGCAACAAGAGTAGAATTAGCAGCAATAGCTTCAGCAAAAAC3′) (SEQ ID NO:10). Underlinedareas represent point-mutations or inserted codons.

A new Sar1(H74L) overexpression plasmid (pPP11) was created bysub-cloning the Sar1 coding region of pLL18 (Phillipson et al., 2001) asa ClaI-XbaI fragment into pLL4, which contains the Cauliflower MosaicVirus 35S promoter, a spacer DNA flanked by ClaI and XbaI sitesoverlapping with the translation intitiation and stop codon, followed bythe 3′ untranslated end of the nopaline synthase gene. An Arf1poverexpressing plasmid pPP5) was created by PCR mediated amplificationof the ARF coding region using the oligonucleotides ARF1-sense(5′GATCACCATGGGGTTGTCATTCGG3′) (SEQ ID NO:11) and ARF1-anti(5′GCTAACTCTAGATCTATGCCTTGCTTGCGAT3′) (SEQ ID NO:12) from first strandcDNA prepared from 5 day old seedlings of Arabidopsis thaliana preparedaccording to established procedures (Denecke et al., 1995). To generateARF1(Q71L), the following two oligonucleotides were used forsite-directed mutagenesis of pPP5: ARF1MS(5′GGGATGTTGGGGGTCTCGACAAGATCCG TCCA3′) (SEQ ID NO:13) and ARF1MA(5′TGGACGGATCTTGTCGAGACCC CCAACATCCC3′) (SEQ ID NO:14) resulting in thederived plasmid pLL20. Underlined regions represent point-mutations. Allconstructions were verified by sequencing analysis. All constructionswere verified by sequencing.

Plant Material and Growth Culture Conditions

Plants (Nicotiana tabacum cv Petit Havana; (Maliga et al., 1973)) weregrown in Murashige and Skoog medium (Murashige and Skoog, 1962) and 2%sucrose in a controlled room at 25° C. with a 16-hours day length at thelight irradiance of 200 μE/m²sec.

Transport Assays

Tobacco leaf protoplasts were electroporated as described (Denecke andVitale, 1995), and plasmid concentrations used are given in the figurelegends. Harvesting of cells and culture medium as well as the enzymaticassays were done as described previously (Denecke and Vitale, 1995;Crofts et al., 1999; Leborgne-Castel et al., 1999). Equal volumes ofcells and medium were loaded on protein gel blots or analysed viaenzymatic assays to determine the fraction of the marker proteins insideand outside of the cells and to calculate the secretion index (Deneckeet al., 1990). Membrane recruitment was assessed by an osmotic shockprocedure described previously (Denecke et al., 1990; Denecke et al.,1992).

Protein Extraction and Enzymatic Assays

Protoplasts were extracted in α-amylase extraction buffer (Crofts etal., 1999) via sonication for 5 seconds. In all cases, extracts werecleared by 10 min centrifugation at 2500 g at 4° C. and the supernatantwas recovered. α-amylase activity was determined as described (Crofts etal., 1999).

Protein Gel Blotting

Samples were loaded after twofold dilution with 2×SDS-PAGE loadingbuffer (200 mM Tris-Cl, pH 8.8, 5 mM EDTA, 1 M sucrose, and 0.1%bromophenol blue). Proteins in SDS-polyacrylamide gels were transferredonto a nitrocellulose membrane and then blocked with PBS, 0.5% Tween 20,and 5% milk powder for 1 hr. The filter was then incubated in blockingbuffer with primary antibody at a dilution of 1/5000 for anti-PATantibodies, anti-c-myc antibodies (Santa Cruz Biotechnology), whereas1/2500 dilutions were used for anti-Sec12 (Bar-Peled and Raikhel, 1997)and anti-Sar1 antibodies (Pimpl et al., 2000). All antisera were fromrabbit and incubation of secondary antibodies and further steps weredone as described (Crofts et al., 1999).

Pulse Labelling and Immunoprecipitation

10⁶ protoplasts were labelled as described previously (Crofts et al.,1998), except that the concentration of labelled methionine was higher(200 μCi/mL). Immunoprecipitation of myc-tagged calreticulinΔHDEL wascarried as described previously (Crofts et al., 1998), but usingpolyclonal anti-c-myc antibodies (Santa Cruz Biotechnology).

Immunocytochemistry

Preparation of ultrathin cryosections from tobacco root tips was carriedout as described previously (Pimpl et al., 2000). Immunogold labellingwas performed with Protein A Sepharose purified barley α-amylaseantiserum diluted 1/100, kindly provided by Birte Svensson (CarlsbergLaboratory, Copenhagen, Denmark). Labelled sections were observed in aPhilips CM10 electron microscope (Philips, Eindhoven) operating at 80kV.

EXAMPLE 1

Partial Secretion of α-Amylase-HDEL is Due to Saturation of the ERRetention Machinery

The addition of an ER retention motif can be deleterious to proteinfunction as it implies a protein modification of the protein itself.Moreover, we demonstrate here that retention is incomplete and subjectto saturation of the receptor. The transport of α-amylase was comparedwith that of the transport of a modified enzyme carrying the ERretention signals HDEL at its C-terminus (α-amylase-HDEL). At differenttime-points after transfection, the culture medium and cells wereharvested and α-amylase activity measured.

FIG. 1A shows that α-amylase was rapidly secreted as judged by the lowintracellular steady state level reached as a result of an equilibriumbetween de novo synthesis and export. Extracellular α-amylase activitywas detected soon after transfection and rose linearly after 6 hours ofexpression. In contrast, α-amylase-HDEL accumulated to high levelswithin the cells and only reached an intracellular steady state level 20hours after transfection. Secretion of x-amylase-HDEL into the mediumbecame apparent after 10 hours of expression. At later time-points, thesecreted levels of the HDEL tagged enzyme increased and the slope of thecurve approached that of α-amylase. At the end of the time course,secretion of both enzymes was thus comparable.

The results demonstrated that the secretion of α-amylase-HDEL was due tosaturation as a result of high intracellular levels. If partialsecretion were due to incomplete presentation of the HDEL motif at theC-terminus of α-amylase, secretion would have occurred immediatelyfollowing synthesis. However, FIG. 1 shows that retention was completeas long as the intracellular levels did not reach a certain threshold.

It should be noted that the total activity obtained was independent ofthe presence of the HDEL motif. This shows that the enzyme was stableand enzymatically active regardless of its position in the secretorypathway. In contrast, the ratio of the extracellular activity to theintracellular activity, termed the secretion index (Denecke et al.,1990), was dramatically decreased by tagging with the retention motif,as shown in FIG. 1B. The secretion index rose linearly over time forboth cargo molecules but was generally an order of magnitude lower forthe HDEL tagged protein as illustrated by the scale of the y-axis. Dueto the linear nature of these curves, the secretion rate of differentcargo molecules can be compared simply via measurement of the secretionindex at a single time-point, conveniently after 24 hours of incubation.

FIG. 2 illustrates that saturation of HDEL-mediated recycling also takesplaces in stably transformed tobacco cell suspensions. α-amylase wassecreted to the culture medium and hardly detectable in the cells.α-amylase-HDEL was detected in the cells, but still efficiently secretedto the culture medium. This illustrates that saturation of the retentionmechanism has taken place.

In conclusion, HDEL tagging is not sufficient to guarantee high fidelityER retention, and saturation can be easily observed if the cargomolecule is stable in the post ER compartments. In case of heterologousprotein expression, saturation of the ER retention mechanism may havebeen difficult to observe due to instability, and could thus be a reasonfor sub-optimal yields.

Saturation of ER Export by α-Amylase Overproduction

To confirm that secretion of HDEL ligands is cargo dosage-dependent andnot due to a change in the physiology of the cells during prolongedincubation, a concentration series of plasmids was used fortransfections and cells were incubated for the constant period of 24hours. FIG. 3A shows that the secretion index for α-amylase-HDEL (blackbars) increased with higher plasmid concentration. Since the incubationtime was identical in all cases, the data are consistent with thesaturation model.

Surprisingly, the control experiment using different concentrations ofthe plasmid encoding α-amylase (white bars) revealed that high dosage ofthe cargo molecule resulted in a reduction of the secretion index,suggesting that saturation of anterograde transport had also occurred.The fact that secretion can be saturated by overexpression suggeststhere is a limiting factor that can be saturated. The data forα-amylase-HDEL (FIG. 3A) are thus the result of superimposing twocounteracting effects, the saturation of anterograde transport and thesaturation of the HDEL receptor. The latter effect appears to be muchstronger, hence the net increase in the secretion index forα-amylase-HDEL.

Differential Effect of Temperature on Anterograde and RetrogradeTransport

To further optimise our transport assay, we tested the influence of thetemperature on the transport of these two cargo molecules. FIG. 3Breveals that the temperature had a strong effect on the total amount ofenzyme activity obtained, but that this effect was identical for bothcargo molecules. In contrast, the secretion index was influenced in adifferential fashion which was dependent on the presence or the absenceof the HDEL motif.

Whereas α-amylase would follow the anterograde transport route, the HDELtagged version will engage in both anterograde and retrograde transportbetween the ER and the Golgi. The results suggest that the efficiency ofretrograde transport increases faster with higher temperature comparedto the efficiency of anterograde transport. The differential effect ofthe temperature on these two molecules illustrates that anterograde andretrograde transport are different cellular processes supported bydifferent molecular machinery.

In addition, there was no sharp low temperature block of secretion, incontrast to findings from mammalian cells, because α-amylase secretionimproved gradually with increasing temperature. The results indicatethat 25° C. may be the optimum temperature for both synthesis andtransport

Sec12p Dosage Dependent Inhibition of α-Amylase and α-Amylase-HDELExport

To show that α-amylase and its HDEL tagged derivative exit the ER inCOPII vesicles, we took advantage of the fact that overproduction ofSec12p reduces ER export (d'Enfert et al., 1991b; d'Enfert et al.,1991a; Hardwick et al., 1992; Barlowe et al., 1993; Barlowe andSchekman, 1993; Nishikawa et al., 1994), presumably via the titration ofSar1 which is essential for COPII vesicle budding (Barlowe et al., 1993;Barlowe and Schekman, 1993; Barlowe et al., 1994). We used theArabidopsis thaliana homologue of Sec12p which was shown to complementthe corresponding yeast mutant (d'Enfert et al., 1992).

FIG. 4 shows that Sec12p overproduction resulted in a clear dosagedependent inhibition of α-amylase secretion. In the case ofα-amylase-HDEL, inhibition of ER export via Sec12p overproduction wouldbe expected to alleviate the saturation of the HDEL receptor as smalleramounts of the ligand reach the cis-Golgi. Indeed, FIG. 4 shows that athigh levels of Sec12 a steeper inhibition of secretion forα-amylase-HDEL was observed, providing additional support for thesaturation model.

Sec12p Overexpression Inhibits COPII Transport via Titration of theGTPase Sar1p

It has been suggested that the ER export inhibition via Sec 12poverexpression is due to titration of the low molecular weight GTPase,Sar1p (d'Enfert et al., 1991b; d'Enfert et al., 1991a; Hardwick et al.,1992; Barlowe et al., 1993; Barlowe and Schekman, 1993; Nishikawa etal., 1994), , thus leading to an inhibition of COPII vesicle buddingwhich is dependent on Sar1 (Barlowe et al., 1993; Barlowe and Schekman,1993; Barlowe et al., 1994). To provide experimental evidence for thisclaim, we attempted to rescue secretion of α-amylase via co-expressionof increasing levels of Sar1p superimposed onto a constant inhibitorylevel of Sec12p. FIG. 5 shows that a partial reconstitution of α-amylasesecretion did occur upon co-expression of Sar1p.

To rule out that this recovery was due to an independent effect of Sar1poverexpression on secretion, increasing levels of Sar1p were tested inthe absence of Sec12p overexpression. In this case, no effect wasobserved on α-amylase secretion (FIG. 6). This result is very importantas it shows that the recovery observed in FIG. 5 must have been due to asuppression of the Sec12p effect, and is not due to an independenteffect of increasing Sar1 levels. This provides final proof of thetitration hypothesis, for which no direct proof existed to date(d'Enfert et al., 1991b; d'Enfert et al., 1991a; Hardwick et al., 1992;Barlowe et al., 1993; Barlowe and Schekman, 1993; Nishikawa et al.,1994).

To demonstrate that Sec12p can physically interact with Sar1p or alterits cellular location, we co-expressed an increasing amount of Sec12pwith a constant amount of Sar1p. FIG. 7 shows that Sar1p is mostlycytosolic when overproduced, but that increasing levels of Sec 12presult in recruitment of the GTPase to the membrane. The fourexperiments shown clearly establish how Sec12 overproduction can be usedin plant cells as an elegant way to inhibit COPII transport.

Inhibition of COPII Transport via Co-Expression of Mutant GTP TrappedSar1

To establish an alternative method to manipulate COPII transport in oursystem, we took advantage of the known trans-dominant negative effect onCOPII vesicle transport by a GTP trapped mutant of Sar1p in vitro and invivo (Saito et al., 1998; Takeuchi et al., 1998; Takeuchi et al., 2000).This mutant is less sensitive to the GTPase activating activity ofSec23, and results in stabilising vesicles in a coated configurationthat is unable to fuse with the target membrane. FIG. 8A shows that incontrast to wild type Sar1p, mutant Sar1p inhibits α-amylase secretionwhen co-expressed. In addition, co-expression of Sec12 with mutant Sar1causes a further reduction in secretion as compared to co-expression ofeither molecules alone, in contrast to wild type Sar1, which alleviatesthe effect of Sec12 overexpression. Both wild type and mutant Sar1 weredetected in comparable levels using protein gel blotting, thus rulingout any artefact due to differences in the expression levels.

Interestingly, the Sar1 mutant caused a reduction in the total yield ofthe secretory marker. This cannot be due to a general toxifying effectof the GTPase mutant, because co-expression of Sec12 with the sameamount of the mutant GTPase restored higher levels of the secretorymarker. This occurred in spite of a further reduction in secretion.Sec12 overproduction alone also inhibits secretion without a significantchange of the total yield. This means that the negative effect of theGTPase mutant on the total α-amylase levels was not due to theinhibition of the secretion process itself.

To further compare the two methods, we tried to establish whether theGTPase mutant inhibits COPII transport through a displacement of thewild type molecule. FIG. 8B shows that even one hundred fold lowerlevels of the mutant molecule as compared to those used in FIG. 8A couldcause efficient inhibition of secretion, albeit with less loss of enzymeyield (data not shown). In contrast, co expression of the wild typeprotein had no significant rescuing effect at stoichiometric levels.Even when the level of wild type protein was increased, a rescuingeffect was hardly noticeable, unless one hundred fold overexpressionrelative to the mutant was implemented. The results clearly illustratethe strong dominant negative effect of this mutant.

Together, the results suggest that the GTPase mutant inhibits the COPIItransport mechanism at a different level compared to Sec 12, which actsthrough a depletion of Sar1. It is likely that Sec 12 overexpressioninhibits the recruitment of the COPII coat which is dependent on Sar1,and thus acts at the earliest possible position in the pathway. Incontrast, the GTPase mutant prevents uncoating of the vesicles and thusacts at a later stage. Both methods are thus independent approaches tomanipulate COPII transport in vivo.

ER Export of a Bulk Flow Marker is COPII Dependent and Results inDegradation

In vitro generated COPII vesicles from yeast have been shown to containanterograde cargo molecules such as yeast α-factor, but not ER residentssuch as BiP (Barlowe et al., 1994). However, bulk flow to the cellsurface has repeatedly been shown to occur for a number of solublepassenger molecules in plant cells (Vitale and Denecke, 1999), albeit atlow rates. This either suggests differences in the early secretorypathway between yeast and plants, or that in addition to COPII vesicles,other transport mechanisms exist to carry bulk flow out of the ER. Todistinguish between these possibilities, we tested whether the secretionof bulk flow markers (Denecke et al., 1990) or ER residents (Crofts etal., 1999) is COPII dependent,

FIG. 9 shows that secretion of the bulk flow marker phosphinothricinacetyl transferase (PAT) (Denecke et al., 1990; Denecke et al., 1992) isinhibited by Sec12p overexpression as well as mutant Sar1 co-expression,demonstrating that PAT was transported in a COPII dependent fashion. Incontrast to α-amylase, the increase in intracellular PAT levels exceededthe reduction in extracellular PAT. This was unexpected and suggeststhat a significant portion of PAT is degraded when allowed to proceedfurther than the ER in the secretory pathway.

ER Export of ER Residents is COPII Dependent and Results in Degradation

To test if ER residents are also transported in a COPII dependentfashion, a truncated form of calreticulin lacking its HDEL signal(calreticulinΔHDEL) (Crofts et al., 1999) was co-expressed with anincreasing amount of Sec12p. FIG. 9 shows that secretion ofcalreticulinΔHDEL is only detected when the molecule is overexpressed,confirming earlier observations (Crofts et al., 1999). However,inhibition of COPII dependent ER export led to a dramatic recovery ofintracellular calreticulinΔHDEL. This is particularly clear at lowexpression levels where no secretion is observed but Sec12 mediatedrecovery of calreticulinΔHDEL is very high. The latter result was evenmore surprising and suggests that most if not all the calreticulinmolecules that escape from the HDEL mediate recycling are in factdegraded instead of transported to the cell surface.

In contrast to FIGS. 4-7, the experiment shown in FIG. 9 was done usinga 48 hour incubation to maximise the effect of the inhibitors. Tocompare the data with those of FIGS. 4-7, we have also conducted thesame experiment with the secretory marker α-amylase (FIG. 7, lowerpanel). Under these conditions, all secretion indices are approximatelytwice as high as in FIG. 4, as predicted from the linear relationship ofthe secretion index seen in FIG. 1. The longer incubation period allowedmore time for Sec12 overproduction or mutant Sar1 production. Thisincreases the sensitivity of the assay, which can be deduced from thesmaller quantities of Sec12 or mutant Sar1 encoding plasmids used. Inaddition, the GTP trapped mutant can also exhibit a dosage dependenteffect and reduces the total yield to a lesser extent when lower plasmidconcentrations are used as compared to those in FIG. 8A. These resultsillustrate the quantitative nature of our transport assays and thatco-expression of the secretory marker can be used as a routine method inconjunction with Sec12 or the Sar1 mutant to document inhibition of ERexport.

Bulk Flow is Efficient and can Lead to Proteolysis

The data in FIG. 9 allow a direct comparison of the intracellularaccumulation of the two test molecules PAT and calreticulinΔHDEL withthat of the secretory marker α-amylase. It is shown that COPII transportinhibition leads to a similar, if not higher accumulation of the twotest molecules. This is particularly clear in the case ofcalreticulinΔHDEL when it is expressed at low levels. Since neitherinhibition strategy has led to an increase in the total yield of thesecretory marker, it can be concluded that the capacity of the ER tosynthesize proteins was not higher. Hence, the increase in the totallevels of PAT and particularly calreticulinΔHDEL cannot be due to ahigher synthesis rate or an indirect effect on the 35S promoter, whichcontrols the transcription of these three cargo molecules.

One farther control experiment was conducted to rule out an increasedsynthesis rate of calreticulinΔHDEL as a result of the inhibition of ERexport. At the intermediate time-point of 24 hours, when transientexpression is still active but Sec12 overexpression already has a strongeffect on the ER export process, we compared the steady state proteinlevels with the protein synthesis rate at that time-point. The formerwill have accumulated over the complete duration of the 24 hour periodand depends on the rate of synthesis as well as the rate of degradation.

To estimate the synthesis rate, we conducted a pulse-labelling of thecells for 30 minutes starting at the 24 hour time-point. Subsequentquantitative immunoprecipitation and SDS-PAGE revealed the amount ofprotein synthesized during this short time-interval and provided anindication of the synthesis rate. FIG. 10 clearly shows that althoughthe steady state protein levels are very much increased by the presenceof Sec12, the amount synthesized during the 30 minute interval (pulse)was almost identical in all cases. This shows once more that Sec12overexpression had no effect on the synthesis of calreticulinΔHDEL, andthat the increase in the steady state level must be the result ofpreventing turn-over.

Comparison of FIGS. 9 & 10 reveals that at 24 hours, the lowest level ofSec12 has no rescuing effect, whereas at 48 hours a weak stabilisationis observed compared to the control. This again shows that longerincubations provide a more sensitive ER export inhibition assay, ascould be predicted from the fact that it requires time to build upSec12p to sufficient levels to titrate Sar1p.

We concluded that PAT and calreticulinΔHDEL are efficiently exportedfrom the ER via COPII mediated transport, followed by degradation in apost-ER compartment. This prevents the molecule from reaching theculture medium and explains previous results on the poor secretion of ERresidents when devoid of their ER retention signal or during saturationof the HDEL pathway. In addition to an explanation, it also offers astrategy to prevent degradation of heterologous proteins expressed inthe secretory pathway of plants, which can lead to significant advancesin biotechnology. FIG. 10 demonstrates that yields can increase fromundetectable to significant levels, which is very attractive indeed froma commercial perspective. FIG. 11 illustrates the principle of example1, which could indeed be of tremendous commercial value.

EXAMPLE 2

Functional Demonstration of BP80 Functions in Planta

The transport pathway of wild type phytepsin is illustrated in FIG. 12and shows that phytepsin follows the normal COPII dependent transportroute out of the ER. We wanted to determine if phytepsin could bind tothe putative vacuolar receptor BP80 (Kirsch et al., 1994). The bindingproperties of phytepsin have only been shown using in vitro bindingstudies using either affinity columns or cross-linking studies.Therefore, we wanted to determine if phytepsin can interact with BP80 invivo, which will begin to demonstrate the biological relevance of thisputative receptor. In order to achieve these goals we took advantage ofthe documented observations that the binding of BP80 can still occur atpH 7.0 even though it is slightly reduced compared to its optimalbinding of pH 6.5 (Kirsch et al., 1994). Secondly, although the removalof the transmembrane domain and cytosolic tail of BP80 results in asecreted protein, these domains are dispensable for ligand binding (Caoet al., 2000). We took advantage of this feature and generated a solublederivative of BP80 either with or without the ER retention motif HDEL.We also incorporated a c-myc tag within the two molecules so that ourintroduced protein could be easily distinguished from any endogenousBP80, which may cross react with the available BP80 antibodies. TheArabidopsis thaliana BP80 isoform with the highest homology to the peaBP80 was chosen for this purpose (Paris et al., 1997). FIG. 13A showsthat BP80-myc is secreted and that HDEL tagging prevents thiscompletely, showing that the c-myc tag is not interfering with thefolding of the modified soluble BP80 molecules and that the HDEL signalis well presented on the surface.

To test if an interaction between phytepsin and BP80 occurs in vivo, weused the following strategy as described in FIGS. 13B-C to retrievephytepsin from the normal route and deviate it towards the retrograderoute back to the ER. We postulated that BP80 binding to its ligand maynot only occur in the TGN, but also in all other Golgi compartments andperhaps even the ER based on findings on the pH dependence of BP80ligand binding (Kirsch et al., 1996). In addition, we created a BP80depleted phenotype by over expressing phytepsin and causing saturationof the pathway. Therefore, if a constant amount of phytepsin atsaturating levels were co-expressed with increasing quantitiesBP80-mycHDEL, any interaction between the molecules would be observed asa reduction in secretion as well as a reduction in processing due to thecontinued recycling of the molecules due to the presence of the HDEL onBP80.

FIG. 14 illustrates that the HDEL tagged BP80 derivative is capable ofretrieving an un-processed form of phytepsin from the exocytic pathwayas concluded by the inhibition of secretion, reduced levels of processed(vacuolar) forms and increased levels of un-processed forms in thecellular fraction. No significant effect was observed for BP80-myclacking the ER retention motif, suggesting that the retrieval was HDELdependent and not due to non-specific aggregation. More importantly,co-expressing of a construct encoding the secretory marker α-amylaserevealed that the interaction between phytepsin and BP80 is specific asneither BP80-myc nor BP80-mycHDEL influenced the secretion of α-amylase.

Therefore, we conclude that BP80-mycHDEL is capable of retrievingproteins destined to the vacuole and could thus form a suitable approachto prevent vacuolar mis-sorting of heterologous proteins as well.Likewise, BP80 could be specifically engineered to reach the cellsurface and carry proteins, normally destined to the lytic vacuole, tothe cell surface for secretion. The re-direction of a sorting receptorto an unusual location is used here as an example to illustrate how thesecretory pathway can be engineered so as to limit proteolysis and leadthe desired protein to the most suitable location for accumulation andsubsequent recovery. This again has tremendous potential for commercialapplications.

EXAMPLE 3

Brefeldin A Inhibits Both Anterograde and Retrograde Transport

It is established that the molecular target of BFA is the guanosinenucleotide exchange factor for ARF1p, resulting in a stabilisation of anabortive complex with the GDP-bound form of ARF1p at the Golgi membrane.To test if inhibition of retrograde and anterograde transport can bedissected, we decided to utilise the secreted cargo molecule α-amylase(amy) and its derivative carrying an ER retention signal (amy-HDEL),which permits a quantitative analysis of transport rates in tobacco leafprotoplasts (FIGS. 1-6). We have shown before that the secretion index(SI), the ratio between extra- and intracellular activities, provides asimple way to compare relative transport rates at a defined time-point.In addition, amy and amy-HDEL have identical specific enzyme activityand stability throughout the secretory pathway. While both molecules areexpected to have identical ER export properties (FIG. 16, route 1), onlyamy-HDEL can undergo retrograde transport from the Golgi (FIG. 16, route2). If saturation of the HDEL receptor occurs, amy-HDEL is expected toreach the cell surface in the same manner as amy (FIG. 16, routes 3 and7). We now tested the influence of different concentrations of BFA onthe transport of either cargo molecules via transient gene expression intobacco protoplasts. FIG. 17 shows that BFA causes aconcentration-dependent inhibition of α-amylase secretion, visible evenat very low concentration of the drug. In contrast, the slow secretionof the HDEL tagged derivative is hardly influenced by the drug, and onlyvisible at high BFA concentrations. Corresponding well with previousfindings (Crofts et al., 1999), BFA treatment causes a reduction in thetotal amount of α-amylase that is produced during the incubation period.This effect is less obvious for amy-HDEL.

If it is postulated that inhibition of ER export is equal for both cargomolecules, the results in FIG. 17 would indicate that BFA inhibits bothanterograde and retrograde transport in the secretory pathway. Sinceamy-HDEL follows both transport routes, it would seem logical that itreacts relatively neutrally to the drug, less is transported to theGolgi (route 1), but less is recycled (route 2), resulting in roughly asimilar release from the cells (routes 3 and 7). In contrast, amy willonly follow the anterograde route, and the effect of inhibiting thisroute is easily detected even at low concentration of the drug.

The experiment shown in FIG. 17 was conducted using a standard transientexpression assay with a 24-hour incubation period. This is a longtime-period and it is possible that prolonged inhibition of therecycling of essential COPII vesicle budding components leads eventuallyto an inhibition of anterograde transport. To test if BFA affectsanterograde transport only indirectly, it should be possible to showthat BFA first causes a transient secretion of amy-HDEL before causing acollapse of the COPII anterograde route. To test this, we have carriedout a transport experiment in which stably transformed tobacco cellswere incubated for different time-periods in the presence of BFA. Underthese circumstances, protoplasts contain a steady state level of amy oramy-HDEL from the very beginning of the experiment. This steady statelevel is very high for amy-HDEL, thus secretion of the cargo into themedium should be detected with high sensitivity if BFA first inhibitsCOPII retrograde transport while COPII anterograde transport is stillefficient. FIG. 18 shows that BFA only inhibits the secretion of amy.However, the slow secretion of amy-HDEL is slightly induced afterprolonged incubation (80 minutes and more) at which inhibition of amysecretion becomes significant. It was not possible to detect a transientinduction of amy-HDEL secretion prior to the inhibition of amysecretion.

Sec12p Overproduction Specifically Inhibits Anterograde Transport fromthe ER

The results obtained in FIGS. 17 and 18 prompted us to revisit ourprevious findings on the inhibition of COPII transport usingoverexpression of the Sec12 gene product and co-expression of atrans-dominant negative mutant of the GTPase Sar1p carrying apoint-mutation (H74L) that interferes with GTP hydrolysis (FIGS. 4-10).Although both approaches inhibited α-amylase secretion to a similarextent, the mechanism of inhibition occurs at different steps of thetransport route. While Sec12p overproduction causes titration of thecytosolic Sar1p and thus inhibits COPII vesicle budding, the Sar1pGTPase mutant would interfere with vesicle uncoating and subsequentfusion with the Golgi. Interference with GTP hydrolysis has also beensuggested to affect cargo loading (Nickel et al., 1998; Pepperkok etal., 2000), but this has not been shown for soluble proteins yet. Wethus wanted to compare the effect of these inhibitors on both amy andamy-HDEL. To extend the available tools, we also generated an equivalenttrans-dominant negative mutant of ARF1p carrying a point mutation(Q71L), which is GTP restricted (Pepperkok et al., 2000) and thusbelongs to a similar class of mutation as the H74L mutant of Sar1p.

We have shown previously that Arabidopsis equivalents of Sec12p andSar1p (d'Enfert et al., 1992) can be detected in electroporated tobaccoprotoplasts and distinguished from the endogenous tobacco gene productswhen expression is high. FIG. 19 shows that over expression ofArabidopsis ARF1p is difficult to detect above endogenous levels,although possible when using high plasmid concentrations. This ispossibly due to the higher degree of conservation of ARF1 compared toSar1 among eukaryotes (Pimpl et al., 2000). Given the fact that only asmall percentage of protoplasts is transfected with plasmids, thedifference seen in FIG. 19 shows that over expression must besignificant in the transfected cells. Interestingly, over expression ofwild type ARF1p does not influence amy-secretion, whereas the GTPrestricted mutant has a very strong inhibitory effect, similar toprevious observations with Sar1p before (Phillipson et al., 2001). Thismeans that ARF1p does not only influence retrograde transport, whichshould not be utilised by amy at all (FIG. 16), but that anterogradetransport is also affected.

FIG. 20 shows the dosage dependent effect of the three effectormolecules on the transport of amy and amy-HDEL. The two GTPase mutantsinhibit amy secretion strongly but have only weak inhibitory effect onthe secretion of amy-HDEL, and thus confer a BFA-like effect. Incontrast, Sec12p overproduction inhibited amy-HDEL secretion at least asmuch or even more than amy secretion, very much unlike the effect ofBFA. These results suggest that Sec12p overproduction inhibits mainlythe anterograde transport route (FIG. 16, route 1) without affecting thecapacity for HDEL-receptor mediated retrieval from the Golgi (FIG. 16,route 2). In contrast, GTPase mutants of Sar1p and ARF1p inhibit both ERexport and retrieval from the Golgi, but are indistinguishable from eachother and both appear to exhibit a BFA-like effect.

Interference with ARF1 Dependent Transport Causes Mis-Sorting of aVascular Protein

The difficulty to separate inhibition of COPI transport from COPIItransport, as exemplified by the drug BFA (FIGS. 2 and 3) or the GTPasemutants of Sar1p and ARF1p (FIG. 20) is either due to the strong linkbetween COPI and COPII transport, or due to multiple roles of ARF1p inGolgi derived transport. Inhibition of amy secretion by BFA orARF1(Q71L)p could also occur distal to the cis-Golgi and represent theintra-Golgi forward transport (FIG. 16, route 3) that has often beenattributed to COPI transport (Pelham and Rothman, 2000). To distinguishbetween these possibilities, we decided to subject a third soluble cargomolecule to the inhibition assays. Barley phytepsin was shown to leavethe ER in a COPII dependent manner as α-amylase (Törmäkangas et al.,2001), but then leaves the Golgi to reach the vacuolar compartments ofthe plant cell (FIG. 16, route 5). The vacuolar form of phytepsin can beeasily distinguished from its precursor in transit through the ER andthe Golgi via the cleavage of the N-terminal propeptide. Moreover,saturation of vacuolar transport leads to mis-sorting to the cellsurface, as seen for amy-HDEL, and under such conditions the unprocessedprecursor is detected in both the cells and the culture medium.

FIG. 21 shows that overproduction of Sec12p leads to an accumulation ofunprocessed precursor in the cells and an inhibition of phytepsinsecretion. Transport to the vacuole is less affected, as seen by amaintained signal of the processed vacuolar form unless highconcentrations of Sec12p encoding plasmids were co-transfected.Co-expression of Sar1(H74L)p leads to a similar effect on secretion buta more noticeable inhibition of vacuolar sorting as estimated by thediminishing signal of the processed form. In sharp contrast,co-expression of ARF1(Q71L)p causes a transient increase in thesecretion of phytepsin, while transport to the vacuole is inhibited andthe precursor accumulates in the cells. This increase in phytepsinsecretion is observed at concentrations of the GTPase mutant at whichα-amylase secretion is significantly inhibited (compare with FIG. 20).However, at higher concentrations of ARF1(Q71L)p, this secretion isabolished, particularly when using plasmid concentrations as in FIG. 19(data not shown). This illustrates that without dosage experiments, theinduced secretion of phytepsin might have gone unnoticed.

These results suggest that ARF1(Q71L)p mediated inhibition of amysecretion can not be due to a general inhibition of anterogradeintra-Golgi transport and from the cis-Golgi to the cell surface (FIG.16, routes 3 and 7). The data can only be explained if transport to thevacuole (route 5) is more sensitive to ARF1(Q71L)p than the anterogradetransport from the ER via the Golgi to the plasma membrane (routes 1, 3and 7), thus allowing faster secretion of phytepsin while secretion ofamy is reduced.

BFA does not Mimic the Effect of ARF1(Q71L)

The data on the influence of ARF1(Q71L)p on the transport of amy andamy-HDEL suggested that the GTPase mutant exhibited a BFA-like effect(FIG. 20). To test if this similarity is also reflected on the sortingof phytepsin, we tested if BFA can induce secretion of this cargomolecule. Using the same experimental conditions as in FIG. 17, we showthat BFA inhibits processing of the phytepsin precursor (FIG. 22).However, unlike the effect of ARF1(Q71L)p it does not cause an increaseof phytepsin precursor secretion. Instead, secretion was inhibited atthe lowest concentration of BFA used. This suggests that ARF1(Q71L)pinterferes with ARF1p mediated transport in a completely differentfashion from BFA. This is not surprising as BFA is expected to inhibitCOPI vesicle formation itself (Chardin and McCormick, 1999; Peyroche etal., 1999), whereas a GTP restricted mutant of ARF1 does not (Nickel etal., 1998; Pepperkok et al., 2000).

ARF1(Q71L) Induced Secretion Occurs Through, Specific Inhibition ofTransport to the Lytic Vacuole.

Plants contain more than one vacuolar compartment which are reached viadifferent pathways dependent on a variety of sorting signals (Hoh etal., 1995; Paris et al., 1996; Neuhaus and Rogers, 1998). Although ithas been shown that barley phytepsin is detected in both lytic andstorage vacuoles (Paris et al., 1996), it is still unclear which type ofvacuolar sorting signal is utilised by phytepsin (Törmäkangas et al.,2001). For this reason, we wanted to test two well established vacuolarsorting signals which are transplantable and can re-direct cargomolecules from the default pathway to vacuolar compartments.

We fused the C-terminal processed fragment of barley lectin (Bednarekand Raikhel, 1991) and the N-terminal propeptide of sweet potatosporamin (Koide et al., 1997) to the C-terminus of α-amylase, which waspreviously shown to maintain enzymatic activity upon fusion of smallpeptides (Crofts et al., 1999). The sporamin propeptide was shown to actas a sequence specific vacuolar sorting signal regardless of itslocation within the protein (Koide et al., 1997) and was predicted toact properly at the C-terminus of α-amylase. In contrast, the barleylectin propeptide has no sequence consensus but must be localisedstrictly at the C-terminus (Dombrowski et al., 1993). Both sortingsignals are expected to target proteins to the same vacuole in tobacco(Schroeder et al., 1993), albeit via different pathways (Matsuoka etal., 1995). The amy fusion with the sporamin propeptide (amy-spo) wouldbe predicted to act as a BP80 ligand (Koide et al., 1997; Matsuoka andNakamura, 1999), whereas the fusion with the barley lectin propeptide(amy-bl) would be transported in a BP80-independent fashion (Matsuoka etal., 1995).

FIG. 23 shows that both C-terminal fusions (amy-spo and amy-bl) areretained in the cells compared to amylase, but that the fusion with thebarley lectin propeptide exhibits more leakage to the medium than thesporamin propeptide fusion. More detailed analysis revealed that the twofusion proteins with wild type propeptides are mainly recovered from thesupernatant of osmotically shocked cells, and also co-purify with thelytic vacuolar marker α-mannosidase, indicating that they residepredominantly in a vacuolar compartment rather than the ER/Golgi (datanot shown).

We could also demonstrate that all fusion proteins exhibited equalspecific enzymatic activities, as predicted from previous studies(Crofts et al., 1999).

Based on the detailed mutational analysis reported for the NPIRL motifillustrating the crucial role of the large alkyl side-chains ofisoleucine and leucine (Matsuoka and Nakamura, 1999), we replaced thesetwo residues by glycine (amy-spoM), yielding NPGRG. FIG. 23 illustratesthat intracellular retention is completely abolished by thismodification and results in normal secretion as seen for unmodified amy.This illustrates the sequence specific nature of this type of sortingsignal (Neuhaus and Rogers, 1998). Addition of double glycines to theC-terminus of the barley lectin propeptide are known to render thissignal non-functional (Dombrowski et al., 1993), and this is alsoconfirmed for the amylase fusion (amy-blGG).

We subsequently tested the behaviour of these two fusion proteins inresponse to co-expression of ARF1(Q71L)p. FIG. 24A shows that thesecretion of amy-spo was stimulated by ARF1(Q71L)p as observed forphytepsin, except that higher concentrations of the effector were neededin comparison with phytepsin (compare with FIG. 21). In contrast,ARF1(Q71L)p continuously inhibits secretion of amy-bl with increaseddosage, as observed for amy and amy HDEL before. FIG. 24B shows thecalculated secretion index which illustrates even better the completelyopposite behaviour of the two cargo molecules. Also the total activityis affected in a differential fashion. Whereas total amy-spo levelsincrease slightly, amy-bl levels drop significantly with increasingARF1(Q71L)p levels. The latter has also been observed for amy andamy-HDEL (data from FIG. 20, not shown).

To test if sequence specific vacuolar sorting per se is required toexhibit the ARF1(Q71L)p-induced secretion, we tested the mutagenisedform of amy-spo containing the mutant motif NPGRG (amy-spoM). FIG. 25Ashows that secretion is no longer stimulated but in fact inhibited bythe ARF1 mutant, as seen for amy and amy-bl before. This confirms thatinduced secretion by the ARF1 mutant is a signature of sequence specificsorting signals, and thus BP80 ligands. This represents a novel tool todistinguish two different types of soluble cargo molecules followingdifferent sorting pathways.

To confirm results from FIG. 22 on phytepsin, showing that BFA does notexhibit an ARF1(Q71L)p-like effect, we tested the influence ofincreasing concentrations of BFA on the transport of amy-spo as well.FIG. 25B clearly shows that BFA does not induce secretion of the cargomolecule. Since phytepsin and amy-spo behave in a similar fashion, theyare expected to follow similar transport routes, which suggests that atleast a portion of phytepsin is transported by BP80. In contrast to BFA,ARF1(Q71L)p offers a unique opportunity to investigate BP80-mediatedvacuolar sorting via a positive assay, the induced secretion ofBP80-ligands. It also offers an excellent method to re-direct proteins,normally destined to the lytic vacuole, from the Golgi apparatus to thecell surface, leading to secretion. Secretion to the culture medium willconstitute an easier production method due to the simple purificationtechnique, and at the same time prevent proteolysis in the lyticvacuole. Such manipulation, which is only possible with theGTP-restricted mutant of ARF1, would thus constitute a major approach toincrease productivity and cost effectiveness of heterologous proteinexpression by the secretory pathway.

REFERENCES

-   Balch, W. E., McCaffery, J. M., Plutner, H., and Farquhar, M. G.    (1994). Vesicular stomatitis virus glycoprotein is sorted and    concentrated during export from the endoplasmic reticulum. Cell 76,    841-852.-   Barlowe, C., and Schekman, R. (1993). SEC12 encodes a    guanine-nucleotide-exchange factor essential for transport vesicle    budding from the ER. Nature 365, 347-349.-   Barlowe, C., d'Enfert, C., and Schekman, R. (1993). Purification and    characterization of SAR1p, a small GTP-binding protein required for    transport vesicle formation from the endoplasmic reticulum. J. Biol.    Chem. 268, 873-879.-   Barlowe, C., Orci, L., Yeung, T., Hosobuchi, A, Hamamoto, S.,    Salama, N., Rexach, M. F., Ravazzola, M., Amherdt, M., and    Schekman, R. (1994). COPII: a membrane coat formed by Sec proteins    that drive vesicle budding from the endoplasmic reticulum. Cell 77,    895-907.-   Bar-Peled, M., and Raikhel, N. V. (1997). Characterization of    AtSEC12 and AtSAR1. Proteins likely involved in endoplasmic    reticulum and Golgi transport. Plant Phys. 114, 315-324.-   Bednarek, S. Y., and Raikhel, N. V. (1991). The barley lectin    carboxyl-terminal propeptide is a vacuolar protein sorting    determinant in plants. Plant Cell 3, 1195-1206.-   Cao, X., Rogers, S. W., Butler, J., Beevers, L., and Rogers, J. C.    (2000). Structural requirements for ligand binding by a probable    plant vacuolar sorting receptor. Plant Cell 12, 493-506.-   Casadaban, M. J., and Cohen, S. N. (1980). Analysis of gene control    signals by DNA fusion and cloning in Escherichia coli. J. Mol. Biol.    138, 179-207.-   Chardin, P., and McCormick, F. (1999). Brefeldin A: the advantage of    being uncompetitive. Cell 97, 153-155.-   Crofts, A. J., Leborgne-Castel, N., Pesca, M., Vitale, A, and    Denecke, J. (1998). BiP and calreticulin form an abundant complex    that is independent of endoplasmic reticulum stress. Plant Cell 10,    813-824.-   Crofts, A. J., Leborgne-Castel, N., Hillmer, S., Robinson, D. G.,    Phillipson, B., Carlsson, L. E., Ashford, D. A., and Denecke, J.    (1999). Saturation of the endoplasmic reticulum retention machinery    reveals anterograde bulk flow. Plant Cell 11, 2233-2248.-   Denecke, J., and Vitale, A. (1995). The use of protoplasts to study    protein synthesis and transport by the plant endomembrane system.    Methods Cell Biol. 50, 335-348.-   Denecke, J., Botterman, J., and Deblaere, R. (1990). Protein    secretion in plant cells can occur via a default pathway. Plant Cell    2, 51-59.-   Denecke, J., De Rycke, R., and Botterman, J. (1992). Plant and    mammalian sorting signals for protein retention in the endoplasmic    reticulum contain a conserved epitope. EMBO J. 11, 2345-2355.-   Denecke, J., Carlsson, L. E., Vidal, S., Hoglund, A. S., Ek, B., van    Zeijl, M. J., Sinjorgo, K. M., and Palva, E. T. (1995). The tobacco    homolog of mammalian calreticulin is present in protein complexes in    vivo. Plant Cell 7, 391-406.-   d'Enfert, C., Gensse, M., and Gaillardin, C. (1992). Fission yeast    and a plant have functional homologues of the Sar1 and Sec12    proteins involved in ER to Golgi traffic in budding yeast. EMBO J.    11, 4205-4211.-   d'Enfert, C., Wuestehube, L. J., Lila, T., and Schekman, R. (1991a).    Sec12p-dependent membrane binding of the small GTP-binding protein    Sar1p promotes formation of transport vesicles from the ER. J. Cell    Biol 114, 663-670.-   d'Enfert, C., Barlowe, C., Nishikawa, S., Nakano, A., and    Schekman, R. (1991b). Structural and functional dissection of a    membrane glycoprotein required for vesicle budding from the    endoplasmic reticulum. Mol. Cell Biol. 11, 5727-5734.-   Dombrowski, J. E., Schroeder, M. R., Bednarek, S. Y., and    Raikhel, N. V. (1993). Determination of the functional elements    within the vacuolar targeting signal of barley lectin. Plant Cell 5,    587-596.-   Hara-Nishimura, I., Shimada, T., Hatano, K., Takeuchi, Y., and    Nishimura, M. (1998). Transport of storage proteins to protein    storage vacuoles is mediated by large precursor-accumulating    vesicles. Plant Cell 10, 325-836.-   Hardwick, K. G., Boothroyd, J. C., Rudner, A. D., and Pelham, H. R.    (1992). Genes that allow yeast cells to grow in the absence of the    HDEL receptor. EMBO J. 11, 4187-4195.-   Hoh, B., Hinz, G., Jeong, B. K., and Robinson, D. G. (1995). Protein    storage vacuoles form de novo during pea cotyledon development. J.    Cell Sci. 108 (Pt 1), 299-310.-   Humair, D., Hernandez Felipe, D., Neuhaus, J. M., and Paris, N.    (2001). Demonstration in yeast of the function of BP-80, a putative    plant vacuolar sorting receptor. Plant Cell 13, 781-792.-   Kirsch, T., Saalbach, G., Raikhel, N. V., and Beevers, L. (1996).    Interaction of a potential vacuolar targeting receptor with amino-    and carboxy-terminal targeting determinants. Plant Phys. 111,    469-474.-   Kirsch, T., Paris, N., Butler, J. M., Beevers, L., and Rogers, J. C.    (1994). Purification and initial characterization of a potential    plant vacuolar targeting receptor. Proc. Natl. Acad. Sci. USA 91,    3403-3407.-   Koide, Y., Hirano, H., Matsuoka, K., and Nakamura, K. (1997). The    N-terminal propeptide of the precursor to sporamin acts as a    vacuole-targeting signal even at the C terminus of the mature part    in tobacco cells. Plant Phys. 114, 863-870.-   Leborgne-Castel, N., Jelitto-Van Dooren, E. P., Crofts, A. J., and    Denecke, J. (1999). Overexpression of BiP in tobacco alleviates    endoplasmic reticulum stress. Plant Cell 11, 459-470,-   Maliga, P., Sz-Breznovits, A., and Marton, L. (1973).    Streptomycin-resistant plants from callus culture of haploid    tobacco. Nat. New Biol. 244, 29-30.-   Matsuoka, K, and Nakamura, K. (1999). Large alkyl side-chains of    isoleucine and leucine in the NPIRL region constitute the core of    the vacuolar sorting determinant of sporamin precursor. Plant Mol.    Biol. 41, 825-835.-   Matsuoka, K., Bassham, D. C., Raikhel, N. V., and Nakamura, K.    (1995). Different sensitivity to wortmannin of two vacuolar sorting    signals indicates the presence of distinct sorting machineries in    tobacco cells. J. Cell Biol. 130, 1307-1318.-   Murashige, R., and Skoog, F. (1962). A revised medium for rapid    growth and bioassays with tobacco tissue cultures. Physiol. Plant.    15, 473-497.-   Neuhaus, J. M., and Rogers, J. C. (1998). Sorting of proteins to    vacuoles in plant cells. Plant Mol. Biol, 38, 127-144.-   Nickel, W., Malsam, J., Gorgas, K., Ravazzola, M., Jenne, N.,    Helms, J. B., and Wieland, F. T. (1998). Uptake by COPI-coated    vesicles of both anterograde and retrograde cargo is inhibited by    GTPgammaS in vitro J. Cell Sci. 111 (Pt 20), 3081-3090.-   Nishikawa, S., Hirata, A., and Nakano, A. (1994). Inhibition of    endoplasmic reticulum (ER)-to-Golgi transport induces relocalization    of binding protein (BiP) within the ER to form the BiP bodies. Mol.    Biol. Cell 5, 1129-1143.-   Nishimura, N., and Balch, W. E. (1997). A di-acidic signal required    for selective export from the endoplasmic reticulum. Science 277,    556-558.-   Paris, N., Stanley, C. M., Jones, R. L., and Rogers, J. C. (1996).    Plant cells contain two functionally distinct vacuolar compartments.    Cell 85, 563-572.-   Paris, N., Rogers, S. W., Jiang, L., Kirsch, T., Beevers, L,    Phillips, T. E., and Rogers, J. C. (1997). Molecular cloning and    further characterization of a probable plant vacuolar sorting    receptor. Plant Phys. 115, 29-39.-   Pelham, H. R., and Rothman, J. E. (2000). The debate about transport    in the Golgi—two sides of the same coin? Cell 102, 713-719.-   Pepperkok, R., Whitney, J. A., Gomez, M., and Kreis, T. E. (2000).    COPI vesicles accumulating in the presence of a GTP restricted arf1    mutant are depleted of anterograde and retrograde cargo. J. Cell    Sci. 113 (Pt 1), 135-144.-   Peyroche, A., Antonny, B., Robineau, S., Acker, J., Cherfils, J.,    and Jackson, C. L. (1999). Brefeldin A acts to stabilize an abortive    ARF-GDP-Sec7 domain protein complex: involvement of specific    residues of the Sec7 domain. Mol. Cell 3, 275-285.-   Phillipson, B. A., Pimpl, P., Crofts, A. J., Taylor, J. P.,    Movafeghi, A., Robinson, D. G., and Denecke, J. (2001). Secretory    bulk flow of soluble proteins is COPII dependent. Plant Cell 13,    2005-2020.-   Pimpl, P., Movafeghi, A., Coughlan, S., Denecke, J., Hillmer, S.,    and Robinson, D. G. (2000). In Situ Localization and in Vitro    Induction of Plant COPI-Coated Vesicles. Plant Cell 12, 2219-2236.-   Saito, Y., Kimura, K., Oka, T., and Nakano, A. (1998). Activities of    mutant Sar1 proteins in guanine nucleotide binding, GTP hydrolysis,    and cell-free transport from the endoplasmic reticulum to the Golgi    apparatus. J. Biochem. 124, 816-823.-   Schroeder, M. R., Borkhsenious, O. N., Matsuoka, K., Nakamura, K.,    and Raikhel, N. V. (1993). Colocalization of barley lectin and    sporamin in vacuoles of transgenic tobacco plants. Plant Phys. 101,    451-458.-   Takeuchi, M., Tada, M., Saito, C., Yashiroda, H., and Nakano, A.    (1998). Isolation of a tobacco cDNA encoding Sar1 GTPase and    analysis of its dominant mutations in vesicular traffic using a    yeast complementation system. Plant Cell Physiol. 39, 590-599.-   Takeuchi, M., Ueda, T., Sato, K, Abe, H., Nagata, T., and    Nakano, A. (2000) A dominant negative mutant of Sar1 GTPase inhibits    protein transport from the endoplasmic reticulum to the Golgi    apparatus in tobacco and Arabidopsis cultured cells. Plant J. 23,    517-525.-   Törmäkangas, K., Hadlington, J. L., Pimpl, P., Hillmer, S.,    Brandizzi, F., Teeri, T. H., and Denecke, J. (2001). A vacuolar    sorting domain may also influence the way in which proteins leave    the endoplasmic reticulum. Plant Cell 13, 2021-2032.-   Toyooka, K., Okamoto, T., and Minamikawa, T. (2000). Mass transport    of proform of a KDEL-tailed cysteine proteinase (SH-EP) to protein    storage vacuoles by endoplasmic reticulum-derived vesicle is    involved in protein mobilization in germinating seeds. J. Cell Biol.    148, 453-463.-   Vitale, A., and Denecke, J. (1999). The endoplasmic    reticulum-gateway of the secretory pathway. Plant Cell 11, 615-628.-   Wieland, F. T., Gleason, M. L., Serafini, T. A., and Rothman, J. E.    (1987). The rate of bulk flow from the endoplasmic reticulum to the    cell surfaces Cell 50, 289-300.

1. A method of increasing heterologous protein production in a cell bycontrolling proteolysis in the post endoplasmic reticulum (ER)compartment of the cell.
 2. A method of increasing heterologous proteinexpression/production in a cell comprising limiting and/or decreasingproteolysis in said cell by: (i) limiting/preventing export of proteinsfrom the ER to the post ER compartment of the cell; (ii) re-directingproteins from the vacuolar sorting route back to the ER; and/or (iii)re-directing proteins from the vacuolar route on towards the cellsurface.
 3. A method according to claim 1, wherein the cell is of plant,fungal, yeast or mammalian origin.
 4. A method according to claim 1,wherein the cell contains heterologous DNA encoding a mammalian serumprotein.
 5. A method according to claim 1, wherein the cell containsheterologous DNA encoding a mammalian encoding a mammalian hormone.
 6. Amethod according to claim 1, wherein proteolysis is controlled bylimiting/preventing export of proteins from the ER by inhibition ofCOPII transport.
 7. A method according to claim 6 wherein inhibition ofCOPII transport is achieved by either of the following methods: (i)inhibition of COPII dependent vesicle budding via overproduction ofSar1-specific guanosine exchange factor; (ii) co-expression of mutantGTPase which is less sensitive to its GTPase activating protein and as aresult is defective in GTP hydrolysis or; (iii) co-expression of mutantGTPase which is less sensitive to its GTPase activating protein and as aresult is defective in GTP hydrolysis.
 8. A method according to claim 7wherein the exchange factor is Sec12p or an isoform thereof.
 9. A methodaccording to claim 7 wherein the mutant GTPase is Sar1 or ARF1 orisoforms thereof.
 10. A method according to claim 1, whereinre-directing proteins from the vacuolar sorting route back to the ER isby co-expression of a modified vacuolar sorting receptor which carriesan engineered retention signal.
 11. A method according to claim 10wherein the receptor is BP80 or VPS10 or close isoforms thereof.
 12. Amethod according to claim 10 wherein the cell is adapted so that theBP80 phenotype is depleted by producing a BP80-mycHDEL receptor.
 13. Amethod according to claim 10 wherein the BP80 receptor is specificallyengineered so as to reach a cell surface with its protein cargo, so thatthe protein is secreted at the cell surface.
 14. A method according toclaim 1, wherein cells containing heterologous DNA are incubated inappropriate fermentation/incubation media for a period to generatesufficient cell mass.
 15. A method according to claim 1, wherein theproteolysis control step is conducted for about 12 to 48 hrs beforeharvesting the heterologous protein.
 16. A method according to claim 1,for use in large scale production of heterologous proteins.
 17. A methodaccording to claim 1, further comprising any one or more of thefollowing steps: (i) harvesting the protein; (ii) purification of theprotein; (iii) modification of the protein; (iv) formulating the proteinin a suitable diluent, carrier or excipient; or (v) lyophilising theprotein.
 18. A method of producing heterologous mammalian proteinscomprising: (i) incubating cells containing heterologous DNA encoding aprotein of choice in appropriate incubation media until sufficient cellmass is generated; (ii) reducing export of proteins from the ER to thepost ER compartment of the cell and/or re-directing proteins from thevacuolar sorting route back to the ER or on towards the cell surfaceand; (iii) harvesting the heterologous protein.
 19. A method accordingto claim 18 further including the step of disposal of cell debris byfermentation to methane and/or incineration of solid waste.
 20. A methodaccording to claim 18 further comprising any one or more of thefollowing steps: (i) harvesting the protein; (ii) purification of theprotein; (iii) modification of the protein; (iv) formulating the proteinin a suitable diluent, carrier or excipient; or lyophilising theprotein.
 21. A method of producing heterologous mammalian serum proteinscomprising: (i) incubating cells containing heterologous DNA encodingthe serum protein of choice in appropriate incubation media untilsufficient cell mass is generated; (ii) reducing export of proteins fromthe ER to the post ER compartment of the cell and/or re-directingproteins from the vacuolar sorting route back to the ER or on towardsthe cell surface and; (iii) harvesting the heterologous serum protein.22. A method according to claim 21 further including the step ofdisposal of cell debris by fermentation to methane and/or incinerationof solid waste.
 23. A method according to claim 21, wherein proteolysisis controlled by limiting/preventing export of proteins from the ER byinhibition of COPII transport.
 24. A heterologous protein productproduced by the method according claim
 18. 25. A cell adapted so thatproteolysis is limited and/or decreased.
 26. A cell adapted by themethod of claim 1 so that proteolysis is limited or decreased.
 27. Themethod according to claim 26 comprising increasing production of aheterologous protein.
 28. A method according to claim 2, wherein thecell is of plant, fungal, yeast or mammalian origin.
 29. A methodaccording to claim 2, wherein the cell contains heterologous DNAencoding a mammalian serum protein.
 30. A method according to claim 2,wherein the cell contains heterologous DNA encoding a mammalian encodinga mammalian hormone.
 31. A method according to claim 2, whereinproteolysis is controlled by limiting/preventing export of proteins fromthe ER by inhibition of COPII transport.
 32. A method according to claim31 wherein inhibition of COPII transport is achieved by either of thefollowing methods: (iv) inhibition of COPII dependent vesicle buddingvia overproduction of Sar1-specific guanosine exchange factor; (v)co-expression of mutant GTPase which is less sensitive to its GTPaseactivating protein and as a result is defective in GTP hydrolysis or;(vi) co-expression of mutant GTPase which is less sensitive to itsGTPase activating protein and as a result is defective in GTPhydrolysis.
 33. A method according to claim 32 wherein the exchangefactor is Sec12p or an isoform thereof.
 34. A method according to claim32 wherein the mutant GTPase is Sar1 or ARF1 or isoforms thereof.
 35. Amethod according to claim 2, wherein re-directing proteins from thevacuolar sorting route back to the ER is by co-expression of a modifiedvacuolar sorting receptor which carries an engineered retention signal.36. A method according to claim 35 wherein the receptor is BP80 or VPS10or close isoforms thereof.
 37. A method according to claim 35, whereinthe cell is adapted so that the BP80 phenotype is depleted by producinga BP80-mycHDEL receptor.
 38. A method according to claim 35, wherein theBP80 receptor is specifically engineered so as to reach a cell surfacewith its protein cargo, so that the protein is secreted at the cellsurface.
 39. A method according to claim 2, wherein cells containingheterologous DNA are incubated in appropriate fermentation/incubationmedia for for a period to generate sufficient cell mass.
 40. A methodaccording to claim 2, wherein the proteolysis control step is conductedfor about 12 to 48 hrs before harvesting the heterologous protein.
 41. Amethod according to claim 2, for use in large scale production ofheterologous proteins.
 42. A method according to claim 2, furthercomprising any one or more of the following steps: (i) harvesting theprotein; (ii) purification of the protein; (iii) modification of theprotein; (iv) formulating the protein in a suitable diluent, carrier orexcipient; or (v) lyophilising the protein.
 43. A heterologous proteinproduct produced by the method according to claim
 21. 44. A cellcomprising heterologous nucleic acids so that proteolysis is limitedand/or decreased.
 45. The cell of claim 44, wherein said heterologousnucleic acids encode a serum protein or a hormone.
 46. A cell adapted bythe method of claim 2 so that proteolysis is limited or decreased. 47.The method according to claim 46 comprising increasing production of aheterologous protein.